EP1257586A2 - Utilisation de resine echangeuse d'ions (epm-7) en tant que support solide pour la synthese de peptides et la chromatographie d'affinite - Google Patents

Utilisation de resine echangeuse d'ions (epm-7) en tant que support solide pour la synthese de peptides et la chromatographie d'affinite

Info

Publication number
EP1257586A2
EP1257586A2 EP00993512A EP00993512A EP1257586A2 EP 1257586 A2 EP1257586 A2 EP 1257586A2 EP 00993512 A EP00993512 A EP 00993512A EP 00993512 A EP00993512 A EP 00993512A EP 1257586 A2 EP1257586 A2 EP 1257586A2
Authority
EP
European Patent Office
Prior art keywords
resin
peptide
deae
sephadex
affinity chromatography
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00993512A
Other languages
German (de)
English (en)
Other versions
EP1257586A4 (fr
Inventor
Clovis Ryuichi Nakaie
Danielle Alvez Ianzer
Eduardo Maffud Cilli
Mauricio Martins Rodrigues
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Conselho Nacional de Desenvolvimento Cientifico e Technologico - CNPQ
Original Assignee
Conselho Nacional de Desenvolvimento Cientifico e Technologico - CNPQ
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Conselho Nacional de Desenvolvimento Cientifico e Technologico - CNPQ filed Critical Conselho Nacional de Desenvolvimento Cientifico e Technologico - CNPQ
Publication of EP1257586A2 publication Critical patent/EP1257586A2/fr
Publication of EP1257586A4 publication Critical patent/EP1257586A4/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/042—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers characterised by the nature of the carrier
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention refers to two additional applications discovered for a known type of anion exchange resin. These two uses are related to the possibility of applying this type of anion exchanger support as the starting polymer for peptide synthesis as well as for affinity chromatography in the latter case the resm would present double chromatographic properties, such as affinity for purification of macromolecules using resin-bound peptide sequence as well as anion exchanger properties given by cationic sites spread throughout the resin matrix
  • the chromatographic resin related to this invention is the DEAE-Sephadex A-5CA, sold worldwide for decades by the Amershan-Pharmacia Biotech (Upsala, Sweden) as a weak anion exchanger resin for column chromatography.
  • This classification is due to the presence of tertiary diethylamine-ethyl (DEAE) groups in its structure which are characterized by having pKa ranging from 9 to 10 (v.g., Analytical Ion-Exchange Procedures in Chemistry and Biology, Khym, J.X., Prentice Hall, Inc., N. J., USA [1974]).
  • the strong anion exchange resins contain instead, quaternary ammonium groups that do not deprotonate regardless the pH of the media.
  • the matrix of the Sephadex-type resin is constituted of a class of carbohydrate - dextran - with variable amount of crosslinkage which defines the exclusion limit of every sub-type of these resms.
  • the second innovative application of the DEAE-SephadexTM is simply a consequence of its potential to work as solid support for peptide synthesis.
  • a desired peptide would be assembled in its structure and the composite peptide-resin obtained would serve for purification of macromoiecules depending upon the affinity between them and the resin-bound peptide sequence. And if desired, this peptide-resin would function simultaneously as anion exchange and affinity resin for column chromatography.
  • DEAE-Sephadex A50 might be tested against specific antibody generated against this dodecapeptide sequence.
  • this assay would be carried out indirectly by calculating the inhibition induced by the addition of the synthesized peptide-resin upon the interaction between the specific antibody for (NANP) 3 segment and a recombinant protein containing this repetitive peptide sequence.
  • this immunological affinity assay was also performed separately with the (NANP) 3 - peptide and with DEAE-Sephadex A50 for comparison with the peptidil-Sephadex A50 in this inhibition test. The results demonstrated a significant decrease in the inhibition of antibody and the recombinant protein interaction when the peptide-resin was added to the reacting
  • the direct removal of peptide chain from the resin is not possible with the reagent K procedure, we decided to assay chemical "linkers" between the peptide chain and the resin structure introduced specifically for facilitating this final cleavage step with reagent K. For instance, if the desired peptide contains carboxamide function at the C-termina!
  • the FMDF linker was used for All synthesis with DEAE-Sephadex A50 resin and the synthesized peptide-linker-resin complex was treated with reagent K and after precipitation with ethyl ether, the peptide was removed from the resin with 5% acetic acid solution. A white and amorphous powder was isolated after lyophylization of the acid extract. The homogeneity and correct composition of the crude peptide were confirmed by analytical methods such as mass spectrometry, high performance liquid chromatography (HPLC) and amino acid analysis The purity of the crude peptide was rather similar to that observed when conventional resins are used and the corresponding HPLC profile is represented in Figure 2.
  • DEAE-Sephadex A50 is the first anion exchange-type resin containing dextran-type matrix that can be used alternatively as solid support for peptide synthesis.
  • Affinity chromatography assay with peptidil-DEAE-Sephadex A5Q This experiment involved initially the synthesis in DEAE-Sephadex A50 resin, of the repetitive (NANP)s sequence, already mentioned as antigenic epitope found in the sporozoite form of Plasmodi ⁇ m falciparum. We decided to acetylate the peptide N- termina! moiety in order to avoid any ion exchange effect during the affinity chromatogram.
  • MAP multiple antigen peptide
  • the MAP strategy as mentioned will be used expecting for enhancement of the antibody interaction with the peptide-resin as a consequence of the presence of four (acetyl-NANP)3 chains in a single molecule linked to the resin.
  • the chemical protocol followed the same applied for AH synthesis but without the FMDF-linker because the objective was to obtain the composite ⁇ [(acetil-NANP)3]4- Eac4-(Lys)2-Lys-DEAE-Sephadex A50 ⁇ resin for affinity study with the antibody generated from the acetil-(NANP)3 sequence.
  • the Figure 5 details the chemical structure of this peptide-resins. Aiso for affinity investigation, DEAE Sephadex A50 and acetyl-(NANP)3 were also comparatively evaluated in the binding experiments with the peptide antibody.
  • the designed immunoiogical experiment encompassed the following steps: the purified recombinant protein containing the repetitive domain (NANP)n of the circunsporozoite form of Plasmodium falciparum (v.g. Cell, 70, 1021-1033, [1992]) was added to Eiisa's plate (200 ng/well) and maintained overnight at room temperature After 3 washes with phosphate buffer containing 0,05% Tween-20, the plates were treated at 37°C with PBS containing 5% disnatured miik and 1 % serum bovine albumin To calculate the degree of interaction of this recombinant protein with its antibody, the plates were treated with a specific monoclonal antibody for the (NANP) 3 sequence obtained according to the reference in Science 228, 1436-1440 (1985), in a final concentration of 20 ng/ml After 2 h at room temperature, the unbound antibodies were washed with PBS-Tween and light and heavy anti-lgG of rat, conjugated to peroxid
  • the following step involved more directly the estimation of the affinity degree between the peptide (NANP) 3 bound to DEAE-Sephadex resin and its monoclonal antibody This estimation (in percentage) was based on the inhibition induced by the peptide- resin against monoclonal antibody and recombinant protein interaction
  • the inhibition assay for the antibody and free (NANP) 3 or bound to DEAE Sephadex was carried out according to the following procedure

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention se rapporte à la possibilité d'utilisation d'une résine échangeuse d'anions DEAE-Sephadex A50TM en tant que support polymère pour la synthèse de peptides et la chromatographie d'affinité. Il s'est avéré possible de synthétiser un octapeptide modèle (angiotensine II) dans cette résine avec la chimie Fmoc et lorsque des groupes de liaison appropriés sont utilisés, il est possible d'obtenir un peptide libre clivé à partir de cette résine et contenant une extrémité C-terminale sous forme carboxylate ou sous forme carboxamide. Du fait de la faisabilité de la synthèse d'une séquence peptidique de part en part, la séquence répétitive (NANP)¿3? trouvée dans l'épitope antigénique de la forme sporozoïte de Plasmodium falciparum liée à la transmission de la malaria a été assemblée dans la résine DEAE-Sephadex A50. Une colonne avec la résine DEAE-Sephadex A50 synthétisée contenant la séquence (NANP)3 a retenu de manière quantitative un anticorps dirigé contre la séquence (NANP)3 qui a ensuite été retiré au moyen d'un processus de lavage classique, ce qui a démontré la faisabilité d'utilisation de cet anion.
EP00993512A 1999-12-22 2000-12-20 Utilisation de resine echangeuse d'ions (epm-7) en tant que support solide pour la synthese de peptides et la chromatographie d'affinite Withdrawn EP1257586A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
BR9906091-4A BR9906091A (pt) 1999-12-22 1999-12-22 Usos de resina de troca aniÈnica (epm-7) como suporte sólido para a sìntese peptìdica e para cromatografia de afinidade
BR9906091 1999-12-22
PCT/BR2000/000160 WO2001046216A2 (fr) 1999-12-22 2000-12-20 Utilisation de resine echangeuse d'ions (epm-7) en tant que support solide pour la synthese de peptides et la chromatographie d'affinite

Publications (2)

Publication Number Publication Date
EP1257586A2 true EP1257586A2 (fr) 2002-11-20
EP1257586A4 EP1257586A4 (fr) 2003-05-28

Family

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Family Applications (1)

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EP00993512A Withdrawn EP1257586A4 (fr) 1999-12-22 2000-12-20 Utilisation de resine echangeuse d'ions (epm-7) en tant que support solide pour la synthese de peptides et la chromatographie d'affinite

Country Status (4)

Country Link
US (1) US20030212254A1 (fr)
EP (1) EP1257586A4 (fr)
BR (1) BR9906091A (fr)
WO (1) WO2001046216A2 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050261475A1 (en) * 2004-02-13 2005-11-24 Harvard Medical School Solid-phase capture-release-tag methods for phosphoproteomic analyses
WO2019101940A1 (fr) * 2017-11-24 2019-05-31 Sulfotools Gmbh Procédé de préparation de peptides
EP3713950A1 (fr) 2017-11-24 2020-09-30 Sulfotools GmbH Procédé de préparation de peptides

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE69432313T2 (de) * 1993-12-08 2003-10-16 Mitsubishi Pharma Corp Neuartiges sythetisches Peptil, diese enthaltende Zusammensetzung und Arznei-Zusammensetzung und Arznei gegen das Atemnotsyndrom

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DZYUBENKO P S ET AL: "SOLID-PHASE PEPTIDE SYNTHESIS IN AQUEOUS MEDIUM USING 2 NITRO-4-SULFOPHENYL ESTERS" BIOORGANICHESKAYA KHIMIYA, vol. 15, no. 5, 1989, pages 704-705, XP009008619 ISSN: 0132-3423 *
See also references of WO0146216A2 *
ZEL'TSER I E ET AL: "USING SEPHADEX LH-20 FOR THE SOLID PHASE SYNTHESIS OF OXYTOCIN" KHIMIYA PRIRODNYKH SOEDINENII (TASHKENT), no. 1, 1989, pages 106-113, XP009008618 ISSN: 0023-1150 *

Also Published As

Publication number Publication date
WO2001046216A2 (fr) 2001-06-28
WO2001046216A3 (fr) 2002-01-17
US20030212254A1 (en) 2003-11-13
BR9906091A (pt) 2002-06-04
EP1257586A4 (fr) 2003-05-28

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